Tissue engineering represents a promising approach to treat a number of cardiovascular problems including atherosclerosis, damaged valves and heart failure. Cultured blood vessels can be made from extracellular matrix alone or with smooth muscle cells embedded in polymer or collagen gel. Development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue-engineered grafts. Endothelial cells attachment and function on cultured blood vessels is limited and the adherent endothelium function in a procoagulant manner. The objective of the proposed research is to test the following four hypotheses: (1) endothelial cells cultured in direct contact with partially differentiated smooth muscle cells leads to improved adhesion and differentiation of endothelial cells, (2) exposure of cocultured endothelial cells and smooth muscle cells to flow and mechanical stretch regulates differentiation of both cell types; (3) coculture and flow-mediated effects on endothelial cell differentiation are due, in part, to nitric oxide generated by the endothelium in response to flow and mechanical stretch; and (4) enhanced endothelial cell adhesion in coculture can be used to improve endothelial cell adhesion and function on implanted tissue-engineered blood vessels. In order to test these hypotheses, we will develop a novel coculture system in which endothelial cells are cultured directly on partially differentiated smooth muscle cells. In contrast, current coculture systems do not use differentiated smooth muscle cells and separate the two cell types. Such a system is now feasible due to advances in our understanding of smooth muscle cell differentiation in vitro. Close apposition of the cells will facilitate cell-cell communication. Specific aims of the project are to: (1) develop an endothelial cell-smooth muscle coculture system, (2) determine the effect of flow and mechanical stretch on endothelial cell and smooth muscle cell function, and (3) apply coculture methodology to seed endothelial cells onto tissue-engineered vascular grafts. Results from this research will provide a new method to culture endothelium that more closely mimics the physical interaction found in vivo. This system is applicable to development of tissue-engineered vascular grafts.